Note: Descriptions are shown in the official language in which they were submitted.
- 21 941 03
POLYPEPTIDES USEFUL AS CALCIUM CHANNEL BLOCKERS
Thls is a dlvlslonal of Canadlan Patent Appllcatlon
Ser. No. 2,015,505 flled Aprll 26, 1990.
The sub~ect matter of thls dlvisional appllcatlon ls
dlrected to polypeptldes found ln the venom of the Aqelenopsls
aperta splder, more speclflcally those polypeptides found ln
Fractlons G, I, H2, J, Ll, L2 and M descrlbed herelnunder and
such polypeptides havlng substantlally the same amino acld
sequences and having substantlally the same calclum channel
blocking actlvltles as those polypeptldes.
The sub~ect matter of the parent appllcatlon was
restrlcted to polyamlnes present in the venom of the same
splder.
Throughout thls speciflcatlon, lt should be
understood, however, that the expression "thls lnventlon" or
the llke encompasses the sub~ect matter of both the parent
appllcatlon and thls dlvlslonal applicatlon.
The polyamlnes and the pharmaceutically-acceptable
salts thereof antagonlze excltatory amlno acld
neurotransmltters, whlch neurotransmltters affect cells
includlng neuronal cells of a varlety of organlsms including
invertebrates and vertebrates. The polypeptides and one of
the polyamlnes and the pharmaceutlcally-acceptable salts
thereof block calclum channels ln cells including neuronal and
64680-788D
21 941 03
la
muscle cells of varlous organlsms lncludlng invertebrates and
vertebrates. Thls lnventlon also relates to the use of such
polyamlnes and thelr salts ln antagonizlng excltatory amlno
acld neurotransml'tters, whlch neurotransmltters affect cells
such as cells ln the nervous system of an organlsm Per se, ln
the treatment of excltatory amlno acld neurotransmltter
medlated dlseases and condltlons ln a mammal and control of
lnvertebrate pests, and to composltlons comprlslng the
polyamlnes and salts thereof. Further, thls lnvention relates
to the use of the polypeptldes and one of the polyamlnes and
thelr salts ln blocklng calclum channels ln cells such as
cells ln the nervous and muscular system of an organlsm E~_
se, ln the treatment of calclum channel medlated dlseases and
condltlons in a mammal, and ln the control of lnvertebrate
pests, and to composltlons comprislng the polypeptldes,
polyamlne and salts thereof.
64680-788D
_ -2- 2194103
It has been reported that the venom of the spider
Agelenopsis aperta contains at least two toxins which
affect calcium currents. Jackson, H., et al., Soc.
Neu. Sci. Abstr. 12:1078 ~1987). Those authors disclose
a toxin, referred to therein as AG2, whlch has a
molecular we~ght of less than 1,000 daltons and appears
to suppress calcium currents in a broad range of tissues.
~urther, Jackson, ~., et al., Soc. Neu. Sci. Ab~tr.
o 12:730 ~1986) report another toxin from Agelenop~is
aperta comprising a component of about 6,000 M.W. That
toxln i9 reported to effect pre~ynaptic blockade of
~ransmission and lt has been suggested that the toxin
block~ calcium channels asso~iated ~ith the release of
lS neurotransmltter.
Compounds which are excitatory amino acid neuro-
transmitter antagoniqts have a variety of utllitles.
Excitatory amino acid neurotransmitter antaqonists can
find clinical application irl the treatment of such
conditions as seizure, stroke, cerebral ischemia,
neuronal degeneration disorders such as Alzheimer's
disease and epilepsy and as psychotherape~tant~, among
others. See Excitatory Amlno Acids ln ~lealth and
~isease, D. Lodge, Ed., John Wiley and Sons Ltd., New
York, NY 198~.
Further, such compounds are useful
~n the study of the physioloqy ~f cells such as neuronal
cells and in the control of invertebrate pests.
Compounds which are calcium antagonists have a
variety of utilities. Calcium antagonists can find
clinical application in the treatment of such conditions
as angina, hypertension, cardiomyopathies, supraven-
tricular arrhythmias, aesophogeal achalasia, premature
21q4103
--3--
labor and Raynaud's disease among others. See W. G.
Nay~er, Calcium Antaqonists, Academic Press, Harcourt
Brace Javarlovich Publishers, New York, tlY 1988.
Further, such compounds are useful in the study of the
physiology of cells such as neuronal and muscle cells
and in the control of invertebrate pests.
This invention concerns polyamines and polypeptides
found to be present in the venom of the Agelenopsis
aperta spider. The polyamines of this invention and
the fractions in which they are present according to
this invention are as follows.
Fraction A' (AGEL 452):
O H
~ N'~~--~~~N'~~~'''N "~~'~~N"'--~~' 'N~_,~~_~NH2
~ OH H
HO
Fraction Al ~AGEL 468):
01~ 0
N~--~--~'~N~--~--~" ~ N,--~_,~~_,N'_~~~,~N~2
OH H ~1
0}~
~raction A2 (AGEL 448):
~~ H HO
N N\,~_~N~ ,N~_~"~H NH2
'~ 4 21 941 03
Fraction Bl (AGEL 505):
HO HO
I H , H H
U N~ ,N~_ ~_~N~,--~_~N~ H2
Fraction B2 lAGEL 5043:
FAB MS: high resolution: 505.3861 calc. for
5C27H48N6~3
Fracticn E (AGEL 489):
H HO H H
N~ N~,_~_~N~ ,N~,~,_~H NH2
The polypeptides of this invention and the fractions in
which they are present according to this invention are
as follows.
Fraction G-
An amino-terminal amino acid sequence comprising:
H2N-glu-lys-gly-leu-pro-glu-gly-ala-glu-cys-asp-
gly-asn-glu-ser-asp-cys-lys-cys-ala-glv-gln-trp-
ile-lys-cys-arg-cys-pro-trp-lys-trp-his-ile-thr-
gly-glu-gly-pro-cys-thr-cys-glu-arg-gly-leu-lys-
lys-thr-cys-ile-ser-lys-leu-ser-asp-pro-asn-arg-
asn-glu-trp-leu-ser-; molecular weight for the
entire polypeptide according to FAB MS: 7267.
- 5 21 941 03
Fraction H2:
An amino-terminal amino acid seauence comprising:
H2N-ala-lys-ala-leu-pro-pro-gly-ser-
val-cys-asp-gly-asn-glu-ser-asp-cys-lys-
cys-tvr-gly-lys-trp-his-lys-cys-
arg-cys-pro-trp-lys-trp-his-phe-thr-
gly-glu-gly-pro-cys-thr-cys-glu-lys-gly-
met-lys-his-thr-cys-ile-thr-lys-leu-his-
cys-pro-asn-lys-ala-glu-trp-gly-leu-asp-trp-;
molecular weight for the entire polypeptide
according to Ion-Spray ~S: 7793.
Fraction I:
H2N-asp-cys-val-gly-glu-ser-gln-gln-
cys-ala-asp-trp-ala-gly-pro-his-cys-cys-
asp-gly-tyr-tyr-cys-thr-cys-arg-tyr-
phe-pro-lys-cys-ile-cys-val-asn-asn-asn-CONH2; FAB
MS: 4158.
Fraction J:
H2N-asp-glu-pro cvs-ile-pro-leu-gly-lys-
0 ser-cys-ser-trp-lvs-ile-gly-thr-pro-tyr-
cys-cys-pro-his-pro-asp-asp-ala-gly-arg-
arg-thr-trp-cys-leu-val-asp-tyr-ser-arg-
phe-val-thr-ile-cys-ser-gly-arg-lys-tyr-CONH2;
molecular weight for the entire polypeptide
according to FAB ~S: 5506.
Fraction Ll:
An amino-terminal amino acid sequence comprising:
H2N-ile-val-gly-gly-lys-thr-ala-lys-phe-
gly-asp-tyr-pro-trp-met-val-ser-ile-
gln-gln-lys-asn-lys-lys-gly-gly-phe-asp-; appro,Yi-
mate molecular weight for the entire poly?eptide
of about 20,000.
- 21 941 03
--6--
Fraction L2:
A polvpeptide obtained as described in Example 9.
Fraction M:
An amino-terminal amino acid sequence comprising:
S H2N-glu-ala-thr-glu-ala-ala-lvs-val-leu-
ser-asn-leu-asp-glu-thr-val-asp-pro-; appro~imate
molecular weight for the entire polypeptide of
about 80,000.
The polyamines of this invention and the pharma-
ceutically-acceptable salts thereof antagonize excitatory
amino acid neurotransmitters, whicn neurotransmitters
affect cells. Thus, said polyamines are useful in
antagonizing said neurotransmit'cers, per se. The poly-
amines of this invention are also useful in the control
of invertebrate pests and in the treatment of diseases
and conditions in a mammal mediated by excitatory amino
acid neurotransmitters. Said polyamines are useful,
- also, as psychotherapeutants for a mammal.
The polyamine Bl, above, and the polypeptides of
this invention ~lock calcium channels in cells. Thus,
said polvamine B1 and polypeptides are useful in
blocking calcium channels in cells, per se. Said
polvamine B1 and said polypeptides are also useful in
the control of inverte~rate pests and in the treatment
2S of diseases and conditions in a mammal mediated by
calcium channel function in cells.
Also within the scope of this invention are polv-
peptides which have substantiall~ the same amino acid
sequence and substantiallv the same calcium channe~
blocking activity as t~ pol~peptides describ~d 2bo~ e .
This invention also concerns pharmaceutical compo-
sitions comprising said polyamines and pol~peptides and
methods of administering said pol,~amines and polypeptides.
2 1 94 1 03
Venom is obtained from the Agelenopls aperta spider
through the process of milklng by electrical stimulation
according to standard methods well known to those skilled ln
the art. It ls preferred that the method employed ls one
whlch safeguards agalnst contamlnatlon of the whole venom by
abdomlnal regurgitant or hemolymph. Such methods are well
known to those ski]led ln the art. The whole venom so
obtained is stored ln a frozen state at about -78OC. until
used for purlfication as described below.
Purlflcatlon of the constltuents from the w}lole
venom is accompllshed by reverse phase hlgh performance llquid
chromatography (HPLC) on a varlety of preparative and semi-
preparatlve columns such as C-4 and C-18 Vydac~ columns
(Ralnln Instrument Co. Inc., Mack Road, Woburn, Massachusetts
01801). Peak detectlorl is carrled out monochromatically at
220-230 nm. Further analysls of the fractions can be
accomplished with, for example, polychrome UV data collected
wlth a Waters* ~90 dio~e array detector (Millipore
Corporation, Waters Chromatography Dlvlslon, 34 Maple Street,
Mllford, Massachusetts 01757). The fractions from the columns
are collected by known methods such as through the use of an
ISCO*~"FOXY" fractlon collector and an ISCO* 2159 peak
detector (ISCO, 4700 Superlor, Llncoln, Nebraska 68504). The
fractions are collected in approprlately sized vessels such as
sterlle polyethylene laboratory-ware. Concentration of the
fractlons ls then accompllshed by lyophlllzatlon from the
*Trade-mark
64680--788D
- 21 941 03
- 7a -
eluant followed by lyophilization from water. Purity of the
resulting constltuent fractions then can be determi.necl by
chromatographlc analysls uslng an analytlcal column wlth a
gradlent system whlch ls more lsocratlc than the system used
ln the flnal purlflcatlon of the fractlons.
64680-788D
-8- 21 941 03
The structures comprised bv the respectite fractions
are determined according to known analytical methods
such as by mass spectrometry and nuclear magnetic reso-
nance. The polypeptides of this invention are sequenced
according to known methods. For example, S-pyridyl-
ethvlation of cystine residues of the polypeptide under
studv can be performed in solution followed by amino
acid sequencing of the polypeptide. One such procedure
for S-pyridylethylation is as follows. About 1 to
0 10 'Jg of polvpeptide is dissolved or diluted in up to
50 ~1 of a buffer prepared by miYing 1 part lM TrisHCl,
pH ~.5 containing 4 mM EDTA and 3 parts 8M guanidine HCl.
Th~n, Z.5 ~1 of 10~ aqueous 2-mercaptoethanol is added
and the mi.Yture is incubated at room temperature in the
dark under argon for two hours. After incubation, 2 ul
of 4-vinylpyridine (fresh reagent stored under argon at
-20~ C.) is added ar.d the mixture is incubated for
another two hours at room temperature in the dark under
argon. The mixture is then desalted, preferably by
chromatographv on a short, reverse phase column. The
recovered alk~lated pol~peptide is then sequenced acco-d-
ing to known methods.
In practicing this invention and employing the
general procedure outlined above, it has been found
that a suitable column for initial fractionation of the
venom is a C-18 Vydac~ 22 mm x 250 mm, 300 ~ pore size,
10 ~ particle size column. That column is eluted at a
flow rate of 15 ml/min. using a linear gradient program
of 95~ 80~ A, 5~ 20~ B ~0 ~ 30 min.] then
80~ 30~ A, 20~ --~ 70~ 8 ~30 55 min.l where
A is 0.1% aqueous trifluoroacetic acid (TFA) and B is
aceton~trile. The fractions are collected as described
above. Ten fractions so obtained, labeled A, B, E, G,
H, I, J, X, L and M, were chosen for further analysis
-9- 21 941 03
and/or purification. Additionally, it has been found
that fractionation of the whole venom using a C-18
Vydac~ 22 mm x 250 mm, 300 A pore size, 10 ~ particle
size column which is eluted at a flow rate of 15
ml/min. using a linear gradient program of S~ 10%
B, 95~ 90% A [0 30 min.] where A and B are as
described above yields, inter alia, a fraction labelled
A' herein. The elution times of the fractions are
given below in Examples l and 2.
Fractions A, B, G, H, I/J and L are subjected to
further purification using a variety of columns and
-gradient programs. The specifics for each subfraction-
ation and the results thereof are given in Examples 3,
4, 6, 7, 8 and 10 below.
As shown in the following Examples, Fractions A',
Al, A2, Bl, B2 and E comprise polyamine compounds.
Those compounds and the fractions in which they are
found are as follows.
Fraction A' (AGEL 452):
2 0 ~ N~--N~--N~~ H NH2
~ OH H
HO
Fraction Al (AGEL 468):
OH O
~N~--N~--N~--N~~ N~~ NH 2
OH H H
OH
'- 21 941 03
--1 o
Fraction A2 (AGEL 448):
OH H HO H
~a n NH2
Fraction Rl (AGEL 505):
HO H HO R H H
~ N~,_~_~N~"~_~N~ ~N~ ~ ~,~N
Fraction B2 ~AGEL 504):
FAB MS: high resolution: 505.3861 calc. for
27 48 6 3
Fraction E (AGEL 4a9):
HO ~ H
N~ ~N N~ ~N~_~H
H
As shown in the following Examples, Fractions
G, H2, I, J, Ll, ~2 and M comprise polypeptides.
Those polypeptides and the fractions in which they are
found are as follows:
Fraction G:
An amino-terminal amino acid sequence comprising:
H2N-glu-lys-gly-leu-pro-glu-gly-ala-glu-cys-asp-
gly-asn-glu-ser-asp-cvs-lys-cys-ala-gly-qln-trp-
ile-lys-cys-arg-cys-pro-trp-lys-trp-his-ile-thr
gly-glu-gly-pro-cys-thr-cys-glu-arg-gly-leu-lys-
-- 21 941 03
lys-thr-cys-ile-ser-lys-leu-ser-asp-pro-asn-arg-
asn-glu-trp-leu-ser-; molecular weight for the
entire polypeptide according to FAB MS: 7267.
Fraction H2:
An amino-terminal amino acid sequence comprising:
H2N-ala-lys-ala-leu-pro-pro-gly-ser-
val-cys-asp-gly-asn-glu-ser-asp-cys-lys-
cys-tyr-gly-lys-trp-his-lys-cys-
arg-cys-pro-trp-lys-trp-his-phe-thr-
gly-glu-gly-pro-cys-thr-cys-glu-lys-gly-
met-lys-his-thr-cys-ile-thr-lys-leu-his-
cys-pro-asn-lys-ala-glu-trp-gly-leu-asp-trp-;
molecular weight for the entire polypeptide
according to Ion-Spray MS: 7793.
Fraction I:
H2N-asp-cys-val-gly-glu-ser-gln-gln-
cys-ala-asp-trp-ala-gly-pro-his-cys-cys-
asp-gly-tyr-tyr-cys-thr-cys-arg-tyr-
phe-pro-lys-cys-ile-cys-val-asn-asn-asn-CONH2; FAB
MS: 4158.
Fraction J:
H2N-asp-glu-pro-cys-ile-pro-leu-gly-lys-
ser-cys-ser-trp-lys-ile-gly-thr-pro-tyr-
cys-cys-pro-his-pro-asp-asp-ala-gly-arg-
arg-thr-trp-cys-leu-val-asp-tyr-ser-arg-
phe-val-thr-ile-cys-ser-qly-arg-lys-tyr-CONH2;
~AB MS: 5506.
-12- ~1 941 03
Fraction L1:
An amino-terminal amino acid sequence comprising:
H2N-ile-val-gly-gly-lys-thr-ala-lys-phe-
gly-asp-tyr-pro-trp-met-val-ser-lle-
gln-gln-lys-asn-lys-lys-gly-gly-phe-asp-; and an
approximate molecular weight for the entire
polypeptide of about 20,000.
~raction L2:
A polypeptide obtained as described in Example 9.~0 Fraction M:
An amino-terminal amino acid sequence comprising:
H2N-glu-ala-thr-glu-ala-ala-lys-val-leu-
ser-asn-leu-asp-glu-thr-val-asp-pro- and an
approximate molecular weight for the entire
polypeptide of about 80,000.
Given the benefit of the disclosure herein with
respect to the compounds present in the fracti~ns of
venom from Agelenopsis aperta, it is now possible to
obtain said compounds bv methods other than through
isolationipurification from whole venom. All such
methods are within the scope of this invention. For
example, the polyam~nes which are comprised by fractions
A', A1, A2, B1, B2 and E can be made directly by syn-
thetic methods. The polypeptides which are compri~ed
' 2' I, J, L1, L2 and rl and for
which the entire amino acid sequence is described can
be produced synthetically bv in vitro protein s~nthesis
according to well known methods. For example, such
p~lypepti~es can be synthesized ufiing an AB~ 430A solid
phase peptide synthesiz~r (Applied Biosystems, Inc.,
850 Lincoln Center Drive, Foster city, Cal~fornia 94404)
employing standard Merrifield chemistry or other solid
phase chemistries well known to those skilled in the
*Trade-mark
.. .. . ..
-13- 21 941 03
art. The polypeptides can also be produced using recom-
binant DNA techniques through the cloning of coding
sequences for the polypeptides or portions thereof.
Those polypeptides for which only part of the amino
acid sequence is known can be cloned, for example,
through the use of hybridization probes which take
advantage of the now known amino acid sequence informa-
tion. A combination of recombinant DNA techniques and
in vitro protein synthesis can also be employed to
produce the polypeptides of this invention.
It is well known in the art that certain amino
acid substitutions can be made in polypeptides which do
not affect, or do not substantially affect, the function
of said polypeptides. The exact substitutions which
are possible vary from polypeptide to polypeptide.
Determination of permissible substitutions is accom-
plished according to procedures well known to those
skilled in the art. Thus, all polypeptides having
substantially the same amin~ acid sequence and substan-
tially the same calcium channel blocking activity arewithin the scope of this invention.
A synthetic scheme for production of certain
polyamines of this invention of the formula
R H HO H H
U N~N N ~N ~ N N~I Z
~lherein R is H or O~ is shown in Reacti~n Schemes A to
D, below.
-
-14- 21 941 03
REACTION SCHEME A
2 2 + NC ~
2 N~'~CN + Br~'~ " " N-BOC
(I) (II)
BOC-N~N~N~CN
H
~III)
[BOC]2o
BOC
BOC-N'--~ ~"N" " " " " N~'~CN
. BOC
(IV)
BOC
2 '~~~~~" N~--'~~ " " N " " " ~N-BOC + NC
BOC
(V)
~ BOC
NC~ ~N~--~N~ --'~N~_--~N-B~C
BOC
(VT )
[BOC]2o
-15- 21 941 03
BOC BOC
N(~ N ~N ~N/ NH-BOC
BOC
(VII )
BOC BOC
2 ~~N ~N ~N~\N_Boc + NC~
. . BOC H
(VIII)
H BOC BOC
NC~N ~N ~N~ N~\NH_Boc
BOC
(IX)
H BOC BOC
2 \~H----N\~ N~~~N~\ N- BOC
BOC
(X)
-- -16- 2194103
REACTION SCHEME B
~;; ~OH
H H
( X I )
[BOCl 2~
BOC
(XI I )
~N
BOC
(XIII )
-17- 21 941 03
REACTION SCHEME C
OH OCH20CH3 ~3C~
[~3 ~3 3
H H
(XVII)
OCH 2OC~ 3
[~3~;3,,~ ~ CH 3
(XVI I)
OCH20CH3 OC~20CH3
~3~N3~ \¢
H H
(XX) (XIX)
OCH 2~CH 3 OCH 2~CH 3
¢OH~N3~oCH2CH=CH2
BOC BOC
(XXII) (XXI)
'~ -18- 2 1 9 4 1 0 3
REACTION SCHEME D
X + XIII or XXII
R ' H H BOC BOC
N--~N ~N ~N ~ BOC H-BOC
BOC
~R' = H (XIV), R' = OCH20CH3 (XXIII) ]
R ' H OH BOC BOC
~¢ N--~N ~N ~BOC NH-BOC
BOC
[R' = H (XV), R' = OCH20CH3 (XXIV) ]
R H OH H H
~N ~N ~N ~N ~H NH2
[R = H (XVI), R -- OH(XXV) ]
R ' H , H H
~N ~N ~N ~N ~H N~I2
O~OH
(XXIV' )
2 1 94 1 03
--1 9--
REACTION SCHEME E
O O
HO ~ OH ~ HO OCH2CH=CH2
(XXVI~
O ~ O
Il 11
~ OH ~ OCH2CH=CH2
CH30CH20 ~ CH30CH20
(XXVIII) (XXVII)
REACTION SCHEME F
OH O OH O
OH ~ OCH2CH=CH2
OH OH
(XXIX)
OC~20CH3 OCH20CH3
O O
OH ~ ~ OCH2CH=CH2
OCH20CH3 OCH20CH3
(XXXI) (XXX)
- 21 941 03
-20-
R~ACTION SCHEME G
X + XXVIII or XXXI
BOC
N'~ ~N'--~--~--~N~--~'~'' HN-BOC
~ BOC BOC
OCH20CH3
(XXXII)
or
OCH20CH3
O BOC
N'~~--~" N~'~_~'~ ~ N'--~_~--~" N~_~--~" N-BOC
BOC BOC
OCH20CH3
(XXXIII)
BOC
N'~~--~'N'--~--~--~N"~--~--~N"~''~~'N~--'--~--'HN-B~C
~ OH BOC BOC
OCH20CH3
(XXXIV)
or
OCH?OCH
O BOC
N'~~-~''N-'~-~-', , ' N-BOC
OH BOC BOC
OCH20CH3
( XXXV )
~21- 21 q41 03
REACTION SCHEME G (Continued)
( XXX IV ) or ( XXXV )
O
~N ~\N~ N N~~ N~~ N~ 2
)~J OH H
HO
(XXXVI )
or
N~--N~--~N ~NH2
~J H OH H H
OH
( XXXV I I )
-22- 21 941 03
REACTION SCHEME H
BOC
VI ~ 2N N--N--~ ' HN-BOC
BOC
( XXXVI I I )
XXXVIII + XXII
OCH20CH3 H H BOC
~N ~N ~N ~N~N- BOC
N ~ BOC H
BOC
- ( XXX IX )
OCH20C~3 H OH BOC
~N--~N--~N~--N- BOC ---
BOC
(XL)
OH H OH H
~N ~N N ~H~N~12
( XLI )
t
OH H OH H
~N~ N~ N ~N~NH2
O~OH
( XL ' )
- 21 941 Q3
-23-
According to Reaction Scheme A, the polyamine
intermediate compound of formula X is prepared through
a sequence of steps beginning with diaminobutane.
Reaction conditions suitable to prepare the intermediate
compound of formula X according to Reaction Scheme A
are given in Example 11, parts A to I. Reaction Scheme B
illustrates a method for the preparation of the inter-
mediate compound of formula XIII. Reaction conditions
suitable to prepare that intermediate according to
Reaction Scheme B are given in Example 11, parts J to
L. Preparation of the intermediate compound of formula
XXI is shown in Reaction Scheme C. Reaction conditions
suitable for the preparation of the compound of formula
XXI according to Reaction Scheme C are given in
Example 12, parts A to F. Preparation of the polyamine
compounds of this invention of the formulae XVI and XXV
is shown in Reaction Scheme D. Reaction conditions
suitable for the coupling of the intermediate compounds
of formulae X and XIII or X and XXIIand the subsequent
preparation of compounds of the formulae XVI and XXV
are given in Example 11, parts M to O and Example 12,
parts G to I.
Reaction Schema E and F illustrate methods for the
preparation of the intermediate compounds XXVIII and
XXXI, respectively. Reactlon conditions suitable to
prepare the intermediate according to Reaction Scheme E
are given in Example 13, parts A to C. Reaction condi-
tions suitable for the preparation of the intermediate
according to Reaction Scheme F are given in Example 14,
parts A to C. Preparation of the polyamine compounds
of this invention of the formulae XXXVI and XXXVII is
shown in Reaction Scheme G. Reaction conditions suit-
able for the coupling of the intermediate compounds of
formula X and XXVIII or X and XXXI and the subsequent
- 21 941 03
-24-
preparation of compounds of the formulae XXXVI and
XXXVII are given in Example 13, parts D to F and
Example 14, parts D to F, respectively.
Reaction Scheme H illustrates a method for the
preparation of the polyamine compound of this invention
of the formula XLI. Reaction conditions suitable for
the preparation of the intermediate compound of formula
XXXVIII, the coupling thereof to the intermediate
compound of formula XXII and the subsequent preparation
of the polyamine compound of this invention of the
formula XLI are shown in Example 15.
The polyamines of this invention reversibly
antagonize excitatory amino acid neurotransmitters,
which neurotransmitters affect cells such as cells in
the nervous system of a variety of organisms including
invertebrates and vertebrates. The term vertebrates as
used throughout is meant to include mammals. The term
invertebrates as used throughout is meant to include,
for example, insects, ectoparasites and endoparasites.
The polyamine of fraction Bl, as described above, also
reversibly blocks calcium channels present in a variety
of cells such as cells in the nervous and muscular,
including cardiovascular, system of invertebrates and
vertebrates.
The ability of the polyamines of the present
invention to antagonize excitatory amino acid neuro-
transmitters is demonstrated by their ability to block
N-methyl-D-aspartic acid-induced (NMDA) elevations of
cGMP in neonatal rat cerebellums accordinq to the follow-
ing pro~edure Cerebellums from ten 8-14 day old Wistar
.
2 1 q4 1 03
-25-
rats are quickly excised and placed in 4~ C. Rrebs/bi-
carbonate buffer, pH 7.4 and then chopped in 0.5 mm x
0.5 mm sections using a McIlwain tissue chopper (The
Nickle Laboratory Engineering Co., Gomshall, Surrey,
England). The resulting pieces of cerebellum are trans-
ferred to 100 ml of ~rebs/bicarbonate buffer at 37~ C.
which is continuously equilibrated with 9S:S O2/CO2.
The pieces of cerebellum are incubated in such a manner
for ninety minutes with three changes of the buffer.
The buffer then is decanted, the tissue centrifuged ~l
min., 320~ r.p.m.) and the tissue resuspended in 20 ml
of the Rrebs/bicarbonate buffer. Then, 250 ~1 aliquots
(approximately 2 mg) are removed and placed in l.S ml
microfuge tubes. To those tubes are added 10 ~l of the
lS compound under study from a stock solution followed by lO
~1 of a 2.5 mM solution of NMDA to start the reaction.
The final NMDA concentration is 100 ~M. Controls do
not have NMDA added. The tubes are incubated for one
minute at 37~ C. in a shaXing water bath and then 7~0
~l of a S0 mM Tris-Cl, SmM EDTA solution is added to
stop the reaction. The tubes are placed immediately in
a boiling water bath for five minutes. The contents of
each tube then are sonicated for lS seconds usinq a
probe sonicator set at power level three. Ten micro-
liters are removed and the protein determined by themethod of Lowry, Anal. Biochem. 100:201-220 (1979~.
The tubes are then centrifuged (5 min., 10,000 xg), 100
~1 of the supernatant is removed and the level of cyclic
GMP (cGMP) is assayed using a New England Nuclear
(Boston, Massachusetts) cGMP RIA assay according to the
method of the supplier. The data is reported as pmole
cGMP generated per mg. protein.
*Trade-mark
- 21 ~41 33
-26-
The polypeptidec of this invention irreversibly
block calcium channels present in a variety of cells
such as cells in the nervous and muscular system of
invertebrates and vertebrates.
The ability of the polyamine of Fraction B, and
the polypeptides of Fractions G, H2, ~3, I, J, K, J'1'
L2 and M to block calcium channels is demonstrated by
the following procedure. Dissociated rat neocortical
neurons are suspended in Krebs bicarbonate buffer,
pH 7. 4 containing 2mM CaC12 and 1% boving serum albumin
I~SA). The neurons are sedimented ~y centrifugation
and resuspended in the came buffer which additionally
contains l~M fura2/AM (Sigma Chem. Co., P.O. Box 14508,
St. Louis, Missouri 63178). The cells are incubated
for 15 minutes at 37~ C., washed and then incubated for
another 15 minutes in the same buffer without fura2/AM.
The cells are then washed in the above buffer now con-
taining 1.~ mM CaC12 and e~uilibrated at room temper-
ature as a concentrated cell suspension for about 5 to
10 minutes. To a quartz cuvette is added 1.2 ml of
prewarmed (37~ C.) BSA-free buffer containing 1.S mM
CaC12 and 10 mM glucose, then 0.3 ml of the concentrated
cell suspension prepared above. The cuvette is placed
in a thermostated ~37~ C.J holder e~uipped with a
magnetic stirrer and the fluorescence is measured with
a fluorescence spectrophotometer such as a Perkin Elme~
650-40 (Perkin Elmer, Wilton, Connecticut 06897). The
fluorescence signal is allowed to stabilize for ab~ut
one minute. Then 1-4 ul of a stock solution of the
compound under study in Krebs bicarbonate buffer at
app_opriate concentrations is added to the cuvette.
Calibration of the fluorescent signals and fura-2 lea~age
~oxrection are performed using the established procedures
of Nemeth, et al., J. Biol. Chem. 262:5188 (1987). At
*Trade-mark
64680-788D
~ ... . .
, .. . . . . , ,, ,, -
~ -27- 21 941 03
the completion of each test, the maximum fluore~cence
value (~max~ is determined by addition of ~onomycin and
the minimum fluorescence value (Fmin) is determined by
the subsequent addition of 5 mM EGTA to chelate calcium.
Employing the foregoing procedure, calcium channel
blocking by a subject compound ~s shown to occur by a
decrease in fluorescence upon addition of the subject
compound.
Also within the scope of this invention are poly-
peptides having substantially the same amino acidseq~ence as the polypeptides of fractions G, H2,
I, J, L1, L2 and M and which have substantially the
same calcium channei blocking activity as said
polypeptides.
The polyamines of this invention are useful in
antagonizing excitatory amino acid neurotransmitters,
per se. As such, the polyamines are also useful in the
control of invertebrate pests and in the treatment of
excitatory amino acid neurotransmitter-mediated diseases
and conditions in a mammal such as seizure, stroke,
cerebial ischemia, neuronal degenerative disorders such
as Alzheimer's disease and epilepsy. Said polyamines
also are useful as psychotherapeutants in a mammal.
Further, the polyamines are useful in the study of the
physiology of cells including, but not limited to,
cells of the nervous system.
The polyamine of ~raction B1 and the polypeptides
of this invention are useful as calcium channel blockers
in cells, per se. As such, these compounds are also
useful in the control of invertebrate pests and in the
treatment of diseases and conditions mediated by calcium
channels function in cells in a mammal such as angina,
hypertension, cardiomyopathies, supraventr-cular
arrhythmias, aesophogeal achalasia, premature labor and
- h4680-78~n
, ~ :
21 941 03
-
-28-
Raynaud's disease. Further, these compounds are useful
in the study of the physiology of cells including, but
not limited to, cells of the nervous and muscular
system.
Also within the scope of this invention are the
pharmaceutically-acceptable salts of the polyamines and
polypeptides of this invention. Such salts are formed
by methods well known to those skilled in the art. For
example, acid addition salts of the polyamines and base
salts of the polypeptides can be prepared according to
conventional methods.
When a polyamine or polypeptide of this ir.vention
is to be administered to a mammal, it can be admin-
istered alone or in combination with pharmaceutically-
acceptable carriers or diluents in a pharmaceuticalcomposition according to standard pharmaceutical prac-
tice. The polyamines and polypeptides can be adminstered
orally or parenterally with the parenteral route of
administration being preferred for the polypeptides.
Parenteral administration includes intravenous, intra-
muscular, intraperitoneal, subcutaneous and topical
administration.
For oral use of a polyamine or polypeptide of this
invention, the compound can be administered, for example,
in the form of tablets or capsules, or as an aqueous
solution or suspension. In the case of tablets for
oral use, carriers which are commonly used include
lactose and corn starch, and lubricating agents, such
as magnesium stearate, are commonly added. ~or oral
administration in capsule form, useful diluents are
lactose and dried corn starch. When aqueous suspensions
are required for oral use, the active inqredient is
combined with emulsifying and suspending agents. If
desired, certain sweetening and/or flavoring agents can
be added.
2 1 94 1 03
-29-
For intramuscular, intraperitoneal, subcutaneous
and intravenous use, sterile solutions of the active
ingredient are usually prepared, and the pH of the
solutions should be suitably adjusted and buffered.
For intravenous use, the total concentration of solutes
should be controlled to render the preparation isotonic.
When a polyamine or polypeptide or salt thereof of
thiC invention is used in a human subject, the daily
dosage will normally be determined by the prescribing
physician. Moreover, the dosage will vary according to
the age, weight and response of the individual patient,
as well as the severity of the patient's symptoms and
the potencv of the particular compound being
administered.
When a polyamine or polypeptide or salt thereof of
this invention is used in control of invertebrate pests,
said compound is administered to said invertebrate
directly or provided to the environment of said inverte-
brate. For example, a compound of this invention can
~e sprayed as a solution onto said invertebrate. The
amount of compound necessary for control of said inverte-
brate will vary according to the invertebrate and envi-
ronmental conditions and will be determined by the
person applying the compound.
When a polyamine or polypeptide or salt thereof of
this invention is used in the physiological study of
cells, said compound is administered to the cells
according to methods well known to those skilled in the
art. For example, said compound can be administered to
cells in an appropriate physiological buffer. An
appropriate concentration of the compounds of this
invention for use in such studies is 100 ~M. However,
21 941 03
-30-
the concentration of said compounds in such studies may
be greater than or much less than 100 ~M. The amount
of the compound administered will be determined by the
person skilled in the art according to well known
methods.
-31- 21 941 03
EXA~PLE 1
Initial fractionation of whole venom
of Agelenopsis aperta
Whole venom of Agelenopsis aperta, obtained from
Natural Product Sciences Inc., Salt Lake City, Utah
84108 and which had been stored in the frozen state at
about -78~ C., was thawed and l0 to 60 ~l amounts
thereof, diluted to 200 ~l, and loaded onto a C-18
Vydac~ (22 mm x 250 mm, 300 A pore size, 10 ~ particle
size) column and eluted using a flow rate of 15 ml/min.
and a solvent system using a linear gradient program of
5% ~ 20~ B, 95~ _ 80% A l0 30 min.] then 20~
70% B, 80~ 30% A [30 55 min.] where A is
0.1% aqueous TFA and B is acetonitrile. Peak detection
was carried out monochromatically at 220-230 nm and
fractions were collected with an ISCO/~FOXY~ fraction
collector and an ISCO 2159 peak detector. Fractions
were collected from 20 minutes to 60 minutes. Based
upon peak detection, the following fractions were
collected:
Fraction Elution Time
A about 21 minutes
B about 22.75 minutes
E about 27.5 minutes
G about 38 minutes
H about 39 minutes
I about 40 minutes
- J about 40 minutes
L about 4 3 minutes
M about 48 . 3 min~ltes
-32- 21 q41 03
EXAMPLE 2
Fractionation of whole venom of Agelenopsis
aperta to obtain Fraction A'
Whole venom of AgelenOpsis aperta was obtained and
loaded onto a C-18 Vydac~ column as described in
Example 1. The column was eluted using a flow rate of
15 ml/min. and a solvent system using a linear gradient
program of 5% 10% B, 95% 90% A [O 30
min.] where A is 0.1% aqueous TFA and B is acetonitrile.
Peak detection and fraction collection were performed
as described in Example 1. Fraction A' which eluted at
about 12.5 minutes was obtained. Fraction A' was then
prepared for spectral analysis by lyophilization from
the eluent followed by lyophilization from distilled
water, according to standard methods.
The structure of the compound which is comprised
by fraction A' was then determined by the use of FAB
MS. The data so obtained and the structure deduced
therefrom are as follows:
A': FAB MS: M/Z 453 (M~l)
Structure: Benzamide, N-(20-amino-4-hydroxy-
4,8,12,17-tetraazeicos-l-yl)-4-hydroxy
O H
'~'--~--~N'--~--~ N~~~--~--~N~_~N~ ~NH2-
~ OH H H
HO
- 21 941 03
-33-
EXAMPLE 3
Subfractionation of fraction A and
determination of structures therein
Fraction A, obtained as described in Example 1,
was loaded onto a C-4 VydacO (10 mm x 250 mm, 300 A
pore size, 5 ~ particle size) column and eluted
therefrom using a flow rate of 4.0 ml/min. and a
solvent system using a linear gradient program of 0%
10% B, lO0~ 90% A [0 30 min.] where A is
0.1% aqueous TFA and B is acetonitrile. Peak detection
was accomplished using a Waters 990 diode array detector
and fraction collection was accomplished as described
in Example 1. Two fractions were obtained as follows:
Fraction Elution Time
Al about 7 minutes
A2 about 9 minutes
Fractions Al and A2 were then prepared for
spectral analysis by lyophilization from the eluent
followed by lyophilization from water, according to
standard methods.
The structure of the compounds which are comprised
by ractions A1 and A2 were then determined by the use
of FAB MS. The data so obtained and structures deduced
therefrom are as follows:
Al
FAB MS:M/Z 469 (M+l), high resolution:
469.3863 calc. for C23H45N6O with 2.0 mm~
error
structure: benzamide, N-(20-amino-4-hydroxy-
4,8,12,17-tetraazeicos-1-yl)-2,5-dihydroxy
34 21 941 03
OH O
N'--~--~'~N"~--~--~U'~~~'"--'N--N~2.
OH H H
OH
A2
FAB MS:M/Z 449 (M+1), 433, 376, 260, 203
Structure: lH-indole-3-acetamide, N-(16-
amino-4-hydroxy-4,8,13-triazahexadec-1-yl)-
4-hydroxy
OH H HO H
~" ~,N~_~_~'~~~--~--~ ~ N2.
EXAMPLE 4
Subfractionation of fraction B and
determination of structures therein
Fraction B, obtained as described in Example 1,
was loaded onto a C-4 Vydaco (22 mm x 250 mm, 300 A
pore size, 10 ~ particle size) column and eluted
off using a flow rate of 15 ml/min. and a solvent
system using a non-linear gradient program of 0%
0% B, 100% ~ 100% A ro ~ s min.], then 0%
10% B, 100% 90% A [5 20 min.3 (Waters curve
1) then 10% 20% B, 90% 80% A [20 30
min.3 (Waters curve 6) then 20% 50% B, 80%
50% A [30 40 min.] (Waters curve 11) where A is
0.1% aqueous TFA and B is acetonitrile. Peak detection
was accomplished using a Waters 990 diode array detec'or
and fraction collection was accomplished as described
in Example 1. Two fractions were obtained as follows:
2 1 ~4 1 03
Fraction Elution Time
B1 about 18.5 minutes
B2 about 21.5 minutes
The fractions were then prepared for spectral
analysis by lyophilization from the eluent followed by
lyophilization from water according to standard
methods.
The structure of the compound which is comprised
by fraction B1 was determined by the use of FAB MS and
the results thereof are as follows:
FAB MS:M/Z 506 (M+l), 489, 461, 433, 378,
333, 260, 231, 215, 203, 155, 119
high resolution: 506.3810 calc. for
C26 48 7 3
Structure: lH-indole-3-acetamide, N-(20-
amino-4-hydroxy-4,8,12,17-tetraazeicos-1-
yl)-4-hydroxy
HO H RO H H H
N~,--~_~N~ ~N~ ~N----~_~,--N~--'~~~N~2.
The molecular weight of the compound which is
comprised by fraction B2 was determined by the use of
EAB MS and the results thereof are as follows:
FAB MS:
high resolution: 505.3861 calc. for
C27 48 6 3-
z 5 EXAMPLE 5
Structure of compound comprised by fraction E
Fraction E, obtained as described in Example 1,
was prepared for spectral analysis by lyophilization
from the eluent followed by lyophilization from water
21 941 03
-36-
according to standard methods. The structure of the
compound comprised by fraction E was determined using
FAB MS, H-NMR and C-NMR. The data so obtained and
the structure deduced therefrom are as follows:
FAB MS:M/Z 4~0 (M+l), 472, 433, 362, 333,
260, 215, 203, 177, 155, 119
high resolution: 490.3860 calc. for
C26H48N702 with 0.6 mm~ error
H-NMR (500 Mhz, d6-DMSO): 10.89 (s, lH),
8.90-7.85 (m, 6H), 7.55 (d, J-8.0 Hz, lH),
7.34 (d, J=8.0 Hz, lH), 7.19 (s, lH), 7.08
(dd, J=8.0 Hz, J=8.0 Hz, lH), 6.98 (dd, J=8.0
Hz, J=8.0 Hz, lH), 3.50 (s, 2H), 3.13 (m,
2H), 3.03-2.82 (m, 18H), 1.88 (m, 6H), 1.78
lS (m, 2H), 1.62 (m, 4H)
C-NMR (125.76 Mhz, d6-DMSO): 170.94,
136.10, 127.17, 123.75, 120.92, 118.67,
118.26, 111.33, 108.97, 57.39, 57.07, 46.12,
46.12, 44.06, 43.89, 43.89, 36.52, 36.22,
32.78, 26.71, 23.79, 3.06, 22.65, 22.65,
22.45
Structure: lH-indole-3-acetamide, N-(20-
amino-4-hydroxy-4,8,12,17-tetraazeicos-1-
yl)
H HO H H H
~ ~ N~ ~N~,_~_~N\,~ '--'NH2.
Alternatively and preferably, whole venom was
fractionated as described in Example 1 except that the
solvent system and linear gradient program used were 0
20~ B, 100% 80% A [0 30 min.] with peak
detection at 220 nm. The fraction which eluted at
about 26.0 minutes was loaded onto a Dynamax Phenyl
7, ~
2194103
-37-
column (4.6 mm x 250 mm, 60 A pore size, 8 u particle
size) and eluted using a flow rate of 1 ml/min. and
isocratic conditions of 10~ B, 90~ A where A and ~ are
as described in Example 1. Peak detection was accom-
plished using a Waters 990 diode array detector
(~=220 nm) and fractions were collected as described in
Example 1. The fraction which eluted at about 55.27
minutes was lyophilized from the eluent followed by
lyophilization from water according to standard methods~0 to yield the compound of this Example.
EXAMPLE 6
Subfractionation of fraction G
Fraction G, obtained as described in Example 1,
was loaded onto a C-18 Vydac~ (22 mm x 250 mm, 300 A
pore size, 10 ~ particle size) column and eluted off
using a flow rate of 10 mltmin. and a solvent system
using a non-linear gradient program of 20~ 30~ B,
80% 70% A [0 40 min.~ (Waters curve 6) and
employing a Waters 990 diode array detector with frac-
tions being collected as described in Example 1.Fraction G, after subfractionation as described above,
eluted from the column at about 22 minutes. That
fraction, which comprises a polypeptide, then was
prepared for sequencing by lyophilization from the
eluent followed by lyophilization from water, accordin~
to well known procedures.
Amino acid analysis o~ the alkylated polypeptide
- of the fraction was obtained using the Waters*Pico-Tag*
method according to manufacturer's specifications.
Sequence data was collected from an Applied Biosystems*
model 470A Protein/Peptide sequence_ (Applied Biosystems,
Inc., 850 Lincoln Center Drive, Foster City, California
94404~ with aqueous TFA conversion. Analysis of the
resulting phenylthiohydantoin amino acids was accom-
plished on line with an Applied Biosystems*model 120APTH analyzer or off line on a DuPont Zorbax*PTH column
* Trade-mark
64680-788D
21 941 03
-38-
(Biomedical Product Department, Chromatographv Products,
E. I. duPont de Nemours and Co., Inc., 1007 Market
Street, Wilmington, Delaware 19898).
As a result of amino acid analysis and FAB MS of
the entire polypeptide it was determined that the
amino-terminal amino acid sequence of part of the poly-
peptide comprised by fraction G was determined as:
H2N-glu-lys-gly-leu-pro-glu-gly-ala-glu-cys-asp-
gly-asn-glu-ser-asp-cys-lys-cys-ala-gly-gln-trp-
ile-lys-cys-arg-cys-pro-trp-lys-trp-his-ile-thr-
gly-glu-gly-pro-cys-thr-cys-glu-arg-gly-leu-lys-
lys-thr-cys-ile-ser-lys-leu-ser-asp-pro-asn-arg-
asn-glu-trp-leu-ser-; and FAB MS: 7267.
EXAMPLE 7
Subfractionation of fraction H and
determination of structures therein
Fraction H, obtained as described in Example 1,
was loaded onto a C-18 Vydaco (22 mm x 250 mm, 300 A
pore size, 10 ~ particle size) column and eluted
off using a flow rate of 10 ml/min. and a solvent system
using a non-linear gradient program of 20% 25% B,
80% ~ 75% A [0 30 min.] (Waters curve 6) then
25% 50% B, 75% 50% A [30 45 min.]
(Waters curve 11), where A is 0.1% TFA and B is
acetonitrile, and employing a Waters 990 diode array
detector with fractions being collected as described in
Example 1. Of the fractions collected, the fractions
of interest were:
Fraction Elution Time
~2 about 36 minutes
H3 about 41 minutes
Fractions H2 and H3, which comprise polypeptides,
then were prepared for sequencing and sequenced
according to the procedures described in Example 6.
- 21 941 03
-39-
The amino terminal amino acid sequence of part of
the polypeptide comprised by fraction H2 was determined
as:
H2N-ala-lys-ala-leu-pro-pro-gly-ser-
val-cys-as~-gly-asn-glu-ser-asp-cys-lys-
cys-tyr-gly-lys-trp-his-lys-cys-
arg-cys-pro-trp-lys-trp-his-phe-thr-
gly-glu-gly-pro-cys-thr-cys-glu-lys-gly-
met-lys-his-thr-cys-ile-thr-lys-leu-his-
cys-pro-asn-lys-ala-glu-trp-gly-leu-asp-trp-;
and the molecular weight of the entire polypeptide
was determined by Ion-Spray MS to equal 7793.
EXAMPLE 8
Subfractionation of fractions I and J
and determination of structures therein
Fractions I and J, obtained as described in
Example 1 and which were not adequately separated due
to t~e similar elution times as shown in Example 1,
were loaded together onto a C-4 Vydaco (22 mm x 250 ~m,
300 A pore size, lO ~ particle size) column and eluted
off using a flow rate of 10 ml/min. and a solvent system
using a non-linear gradient program of 20~ 30% B,
80% 70% A [0 30 min.] (Waters curve 6), then
30% 50% B, 70% 50% A [30 45 min.]
(Waters curve 11) where A is 0.1% aqueous TFA and B is
acetonitrile. Peak detection and fraction collection
were accomplished according to the procedures described
in Example 6. Of the fractions collected, the
fractions of interest were:
- 21 941 03
-40-
Fraction Elution Time
I about 22 minutes
J about 27.5 minutes
Fractions I and J, which comprise polypeptides,
were prepared for sequencing and sequenced according to
the procedures described in Example 6.
The amino acid sequence and FAB MS of the poly-
peptide comprised by fraction I were determined as:
H2N-asp-cys-val-gly-glu-ser-gln-gln-
cys-ala-asp-trp-ala-gly-pro-his-cys-cys-
asp-gly-tyr-tyr-cys-thr-cys-arg-tyr-
phe-pro-lys-cys-ile-cys-val-asn-asn-asn-CONH2 and
FAB MS: 4158.
The amino-terminal amino acid sequence of part of
the polypeptide and FAB MS of the entire polypeptide
comprised by fraction J was determined as:
H2N-asp-glu-pro-cys-ile-pro-leu-gly-lys-
ser-cys-ser-trp-lys-ile-gly-thr-pro-tyr-
cys-cys-pro-his-pro-asp-asp-ala-gly-arg-
arg-thr-trp-cys-leu-val-asp-tyr-ser-arg-
phe-val-thr-ile-cys-ser-gly-arg-lys-tyr-CONH2;
and FAB MS: 5506.
- 21 941 03
-41-
EXAMPLE 9
Subfractionation of fraction L and
determination of structures therein
Fraction L, obtained as described in Example 1,
S was loaded onto a C-18 Vydaco (lO mm x 250 mm, 300 A
pore size, S ~ particle size) column and eluted
off using a flow rate of 3.5 ml/min. and a ~olvent
system using a linear gradient program of 25% _ 40
B, 75~ 60~ A [0 30 min.] where A is 0.1~
aqueous TFA and B is acetonitrile. ~eak detection and
fraction collection were accomplished according to the
procedures described in Example 6. Of the fractions
collected, the fractions of interest were:
Fraction Elution Time
Ll about 20.25 minutes
L2 about 22.5 minutes
Fractions Ll and L2, which comprise polypeptides,
were prepared for sequencing and sequenced according to
the procedures described in Example 6.
The amino-terminal amino acid sequence of part of
the polypept~de comprised by fraction Ll was determined
as:
112N-ile-val-gly-gly-lys-thr-ala-lys-phe-
glv-asp-tyr-pro-trp-met-val-ser-ile-
gln-gln-lys-asn-lys-lys-gly-gly-phe-asp-
and the molecular weight of the entire polypeptide was
determined by gel electrophoresis according to ~nown
methods to be about 20,000.
EXAMPLE 10
Determination of structure of
compound comprising fraction M
Fraction M, obtained as described in Example 1 and
which comprises a polypeptide, was prepared for
sequencing and sequenced according to the procedures
described in Example 6.
2194103
-42-
The amino-terminal amino acid sequence of part of
the polypeptide comprised by fraction M was determined
as:
H2N-glu-ala-thr-glU-ala-ala-lys-val-leu-
S ser-asn-leu-asp-glu-thr-val-asp-pro-
and the molecular weight of the entire polypeptide was
determined by gel electrophoresis according to known
methods to be about 80,000.
EXAMPLE 11
lH-Indole-3-acetamide, N-(20-amino-4-
hydroxy-4,8,12,17-tetraazeicos-1-yl
Svnthesis of the title compound, ascertained to be
comprised by fraction E as described in Example 4, was
accomplished as described below.
A. ~2N ~ 2 + NC
H2~N~CN
(I)
Under nitrogen atmosphere, 13.22 g (0.15 mole)
diaminobutane and 3.5 ml methanol were stirred and
9.94 ml (0.15 mole) of acrylonitrile was added via a
syringe pump over a two hour period with cooling to
0-5~ C. The mixture, after about 18 hours, was chro-
matographed on 600 g silica gel using a solvent system
of 3:1 CH2C12/MeOH (2 liters) followed by 3:1 CH2C12/MeOH
containing 5~ by volume isopropylamine (2 liters). The
fractions containing the coupled product were concen-
trated by evaporation in vacuo and vielded a vellow
viscous oil. NMR analysis verified the product to be
the coupled product of formula (I) shown above.
- 21 941 03
-43-
72222-144
H
B. H2 ~ N~,~CN + Br~'~'~N-BOC
(I) (II)
BOC-N'--'--~'~N~'~~~_~'N'~'~CN
H
(III)
Under nitrogen atmosphere, 5.78 g ~0.041 mole) of
compound of formula I, prepared as described in part A
above, was added to 75 ml of CH3CN and 11.48 g of KF
Celite*and stirred. To the stirring mixture was added
a solution of 9.75 g (0.041 mole) of compound of
formula II, prepared according to the procedure
described in Preparation A, in 25 ml of CH3CN. The
reaction was heated to reflux and monitored by TLC (3:1
CH2C12/MeOH). After three hours, the reaction was
allowed to cool and stand at room temperature. The
Celite was then filtered off and the filter-cake was
washed well with dichloromethane. The filtrate was
concentrated _ vacuo. The crude product contained in
the concentrated filtrate was chromatographed on silica
gel using 2 liters of 3:1 CH2C12/MeOH, then 2 liters of
3:1 CH2C12/MeOH and 10 ml isopropylamine, then 2 liters
of 3:1 CH2C12/MeOH and 30 ml isopropylamine. The
product eluted from the column after the column became
saturated with isopropylamine but was still impure.
All product fractions were combined and concentrated.
Silica gel was prepared by slurrving 500 g of silica
gel in CH2C12 and 125 ml isopropylamine and then used
to pack a column in the standard manner. The crude
product was loaded onto the column using dichloro-
methane. Then, 500 ml of dichloromethane was run
through the column, followed by one liter of 3:1
CH2C12/MeOH. The product fractions were combined
*Trade-mark
2 1 94 1 03
and concentrated ~n vacuo to yield 1.5 g of product of
formula III, above. Another 2 g of product of
formula III was eluted off the column using one liter
of 3:1 CH2Cl2/MeOH and 30 ml isopropylamine.
C. III ~ [BOC~2o
BOC
BOC-N~N~N~CN
BOC
(IV)
Under nitrogen atmosphere, 3.5 g (11.8 mmoles) of
compound of formula III, prepared as described in
part B, above, and 150 ml dichloromethane were stirred
and 5.2 g (23.6 mmoles) of di-t-butyldicarbonate were
added. The mixture was stirred for approximately 63
hours at room temperature. The mixture was then concen-
trated in vacuo and chromatographed using 400 g of
silica gel and a 60:40 hexane/ethyl acetate solvent to
vield 5.62 g of compound of formula IV as an oil.
D. BOC
NC"~,~N~_~"~" N~'~'~N-BOC
BOC H
(IV)
- BOC
2 '----''~--'N~_,~~_~~~~~_~'~N-BOC
BOC H
(V)
Under nitrogen atmosphere, 3.0 g of compound of
formula IV, prepared as described in part C, above, was
dissolved in 30 ml of acetic acid in a 250 ml Parr
bottle. To that solution was added 3.0 g of
Pd(OH)2/carbon and the mixture was hvdrogenated at
50 p.s.i. H2 pressure for 2 hours. The catalyst was
2l 941 03
-45-
removed by filtration and the cake was washed well with
acetic acid. The filtrate was concentrated, taken up
in 75 ml of dichloromethane, washed twice with 75 ml of
lN NaOH and dried over X2CO3. The solution was concen-
trated in vacuo to yield 2.86 g of product of formula Vabove.
E. V + NC ~ .
H BOC
NC "~,,N~_,~" N~ ,~N,'~_~ N-BOC
BOC
(VI)
Under nitrogen atmosphere, 2.86 g (5.7 mmoles) of
compound of formula V, prepared as described in part D,
above, was dissolved in 75 ml methanol. With stirring,
O.41 ml (6.3 mmoles) of acrylonitrile was added to the
mixture and the reaction was stirred overnight. The
reaction mixture was then concentrated, reconcentrated
lS three times from 30 ml dichloromethane and stripped of
solvent in vacuo to yield 3.18 g of compound of
formula VI as an oil.
F. BOC BOC
VI NC~ N~'~,'N~~~_~~~N~--~_~'~ N-BOC
BOC H
(VII)
Under nitrogen atmosphere, 3.18 g (5.7 mmoles) of
compound of formula VI, prepared as described in part E,
above, was combined with 100 ml of dichloromethane and
stirred into solution. To that solution was added
1.37 g (6.3 mmoles) of di-t-butyldicarbonate and the
reaction was stirred for 90 minutes at room temperature.
The reaction mixture was then concentrated and chromato-
graphed on 300 g silica gel using 60:40 hexane/ethyl
acetate as the eluent. The product fractions were
21 941 03
-46-
combined, concentrated, extracted with 3 x 20 ml
dichloromethane and stripped of solvent in vacuo to
yield 3.12 g of product of formula VII, above.
G.BOC BOC
VII H2N~ ~N~_~~~,--N\_~~~_~~~N--~~_~'~N-BOC
BOC
fVIII)
S Following the procedure described in part D,
above, 3.12 g (4.76 mmoles) of compound of formula VII,
prepared as described in part F, above, was dissolved
in 30 ml acetic acid and hydrogenated at 55 p.s.i. for
2 hours in the presence of 3.0 g Pd(OH)2/carbon to
yield 3.02 g of product of formula VIII, above, as an
oil.
H. VIII + NC ~
H BOC BOC
NC~--~,--N~_~ ~" N~ " N~_~~~_~~~--~~'~ N-BOC
BOC
(IX)
Under nitrogen atmosphere, 1.0 g (l.S mmoles) of
compound of formula VIII, prepared as described in
part G, was dissolved in 10 ml methanol. To that
solution was added 0.1 ml (1.67 mmoles) of acrylonitrile
and the reaction was stirred overnight. Then, the
reaction mixture was concentrated, reconcentrated three
times from 20 ml of dichloromethane and stripped of
solvent in vacuo to yield 1.0 g of product of formula IX,
above.
-47- 21 941 03
I. IX
BOC BOC
H
H2N N~ ,N~_" N~ ,~, N-BOC
BOC
(X)
Following the procedure described in part D,
above, 1.0 g (1.4 mmoles) of compound of formula IX,
S prepared as described in part H, above, was dissolved
in 30 ml acetic acid and hydrogenated at 50 p.s.i. for
2.5 hours to yield 0.85 g of product of the formula X,
above.
J. ~ H ~ O
H H
(XI)
A solution containing 6.78 g (20 mmoles) tetrabutyl-
ammonium hydrogen sulfate and 75 ml H2O was prepared by
stirring and 3. 36 g (40 mmoles) o. NaHCO3 was added as
foaming would allow. Then, 3.5 g (20 mmoles) of indole
acetic acid was added, the mixture was stirred for 5
minutes and 75 ml of CHC13 was added. The mixture was
stirred for another 5 minutes and the bottom two layers
were separated. The aqueous layer was salted with
Na2SO4 and extracted with CHC13. All CHC13 extracts
were combined and dried over Na2SO4. The resulting
clear amber colored solution was concentrated, chased
with 3 x 75 ml acetone and finally dissolved in 75 ml
acetone. Then, 1.9 ml (22 mmoles) of allyl bromide was
added and the mixture was allowed to stand for 30 minutes.
The mixture was then concentrated and chromatographed
21 941 Q3
-48-
on silica gel using 3:1 hexane/ethyl acetate to yield
3.57 g of product of formula XI, above, as a light
vellow oil.
XI
BOC
(XII)
Following the procedure of Angew. Chem. Int.
Ed. Engl. 23:298 (1984), 2.15 g (10 mmoles) of compound
of formula XI, prepared as described in part J, above,
was combined with 20 ml acetonitrile and 2.61 g (12
mmoles) of di-t-butyldicarbonate was added to the
mixture. Then, 0.122 g (1 mmole)-of 4-(N,N-dimethyl-
amino)pyridine was added and the reaction permitted to
proceed for 15 minutes. The mixture was then diluted
out to 125 ml with ethyl acetate, washed 2 x dilute
HCl, 3 x 25 ml H2O, 1 x brine, dried and concentrated.
The product was purified by chromatography on silica
gel using 14.5:1 hexane/ethyl acetate as the eluent to
yield 2.87 g of compound of the formula XII, above, as
a colorless oil.
L. XII ~ ~ OH
BOC
(XIII)
To 3.27 g (10.4 mmoles) of compound of formula XII,
prepared according to part K, above, in 20 ml dichloro-
methane was added 7.4 ml of sodium 2-ethylhexanoate in
ethyl acetate (0.231 g/ml). Then, 0.5 g 03P and 0.5 g
(03P)4Pd were added and the mixture was stirred for 60
minutes. The mixture was then concentrated, ta~en up
in 150 ml ethyl acetate and washed 5 x 25 ml H2O. The
2194103
-49-
aqueous extracts were combined and back extracted with
1 x 25 ml ethyl acetate and 1 x 25 ml diethyl ether.
To the aqueous layer was added 75 ml of fresh ethyl
acetate and then acidified to pH 3. The ethyl acetate
S layer was separated and the aqueous layer was extracted
with 1 x 50 ml fresh ethyl acetate. The tw~ ethyl
acetate extracts were combined and washed with 2
x 20 ml H2O, then l x 20 ml brine, dried, concentrated,
chased with 3 x 30 ml hexane where crvstals formed
during the second chase. The mixture was diluted out
to 75 ml with hexane. The solids were filtered, washed
well with hexane and air dried to yield 1.37 g of white
solids. NMR confirmed the title structure of
formula XIII.
M. XIII + X
H H BOC BOC
N ~N ,~i N ~N N- BOC
BOC
(XIV)
Under nitrogen atmosphere, 200 mg (.73 mmoles) of the
compound of formula XIII was combined with 25 ml
dichloromethane, 84 mg (0.73 mmoles) N-hydroxysuccinimide
and 150 mg (0.73 mmoles) dicyclohexylcarbodiimide where-
upon a precipitate formed almost immediately. The
mixt~re was stirred 3 hours. Then, the dicyclohexylurea
was filtered off and to the filtrate was added dropwise a
40 ml solution of compound of the formula X, prepared as
descri~ed in Example 11, Parts A-I in dichloromethane.
The mixture was stirred for 12 hours then washed with 0.1
NaOH and dried over K2CO3, concentrated and chromato-
graphed using lS0 g silica gel and eluting with 9:1
21 941 03
--s o--
dichloromethane/methanol (0.5 liters) to remove impuri-
ties then with 9:1:0.1 dichloromethane/methanol/iso-
propylamine (2 liters). The product was then concen-
trated, chased with dichloromethane and further concen-
trated to yield 400 mg of the compound of formula XIV,confirmed by 300 MHz NMR.
N. XIV
H OH BOCBOC
N~ ~ ,N~ N-BOC
BOC H
BOC
(XV)
Under nitrogen atmosphere, 0.34 g (0.35 mmoles) of
the compound of formula XIV, prepared as described
above, was combined with 30 ml acetone, stirred into
solution and cooled to about 0~ C. in an ice/acetone
bath. Then, 0.212 g (1.05 mmoles) of 85% 3-chloroperoxy-
benzoic acid in 20 ml of acetone was added dropwise
lS over 8-10 minutes. The reaction was stirred for 30
minutes then diluted out to 150 ml with diethylether
and washed with 2 x 25 ml 10% K2CO3, 2 x 25 ml H2O, 2 x
25 ml 10~ ~2CO3, 2 x 25 ml ~2~ and dried over K2CO3.
The mixture was concentrated, chased with 2 x 30 ml
dichloromethane and concentrated in vacuo to a steady
weight of 0.33 g. NMR analysis showed the product to
be a mixture of the desired product of formula XV,
starting material and by-product. The product mixture
was added to 3 ml of acetic acid to which was added
20 mg NaCNBH3. The mixture was stirred overnight,
stripped of acetic acid in vacuo, dissolved in 50 ml
dichloromethane and washed 1 x 75 ml with aqueous
buffer, pH 7. If necessarv, the pH was adjusted to
' - 21 941 03
--s 1
pH 7 with lN NaOH and the dichloromethane layer was
separated and washed with 1 x 50 ml with aqueous buffer,
pH 7 and l x 25 ml brine, dried over Na2SO4, and concen-
trated in vacuo to a steady weight of 0.31 g. The
S resulting product was chromatographed on 100 g silica
gel using 95:5 ethyl acetate/methanol to elute and
collecting 25 ml fractions. The purified product of
formula XV came off in cuts 18-25. Those cuts were
combined, concentrated, chased with 2 x 5 ml of dichloro-
methane and concentrated in vacuo to a white foam of68 mg which was stored at -80~ C. Cuts 26-40 which
also contained the desired product also contained some
impurity. Cuts 26-40 were combined, concentrated,
chased with dichloromethane and concentrated in vacuo
to a steady weight of 66 mg. The latter product was
combined with 6 mg of the former product which had been
used for NMR studies and with a mixture which had been
prepared according to the procedure described above.
The resulti~g mixture was chromatographed on 75 g silica
gel using 9:S ethyl acetate/methanol to elute and collect-
ing 25 ml fractions. Purified product of formula XV
was present in cuts 16-21. Those cuts were combined,
concentrated, chased with 2 x 5 ml dichloromethane and
concentrated in vacuo to a steady weight of 46 mg which
was stored at -80~ C.
O. . XV
H ~~ H H
N~ ~N\~ ,N~ ,N~_~ ~ H2
(XVI)
-52- 2 1 94 1 03
Under nitrogen atmoqphere~ loa mg (0.109 mmoles)
of compound of the formula XV, prepared as described
above, was dissolved in 1.5 ml dichloromethane. Then,
2 ml of trifluoroacetic acid was added and the mixture
was stirred for one hour at room temperature. The
reaction mixture was then concentrated to a fiLm and
15 ml diethylether was added. The film turned to solid
and, after 30 minutes of stirring, vielded white powder.
The powder was filtered, washed well with diethylether,
dried under nitroqen, then evacuated to a steady weight
of 109 mg of the title compound of this Example.
EX~MPLE 12
lH-Indole-3-acetamide, N-(20-amino-4-hydroxy-
4,8,12,17-tetraazeicos-1-yl)-4-hydroxy
Synthesis of the title compound, ascertained to be
comprised by Fraction Bl as described in Example 3, was
accomplished as described below.
OH OCH20CH3
H R
~XVII)
Under argon atmosphere using flame dried glassware,
4.0 g (30 mmoles) of 4-hydroxyindole was combined with
25 ml of Aldrich dry DMF and stirred into solution.
Then, 1. 44 g (30 mmoles) of NaH as a 50~ oil dispersion
was added. After foaming had subsided, 2.5 ml of
chloromethylmethyl ether (Aldrich*) was added and the
resulting dark green solution was stirred overnight.
Then, the solution was diluted to 125 ml with ethyl
acetate, washed with 5 x 25 ml H2O and 1 x 25 ml brine,
dried over Na2S04 and chromatographed using 4:1
hexane/ethyl acetate. The product containing fractions
*Trade-mark
21 941 03
-53-
were combined and concentrated to yield 2.70 g of
compound XVII as an oil.
2 3
XVII + ~N~ N~
(XVIII)
In a 50 ml, 3 neck round bottom flask equipped
with a mechanical stirrer and a nitrogen atmosphere was
cooled 1.3 g of aqueous formaldehyde (37%) and 2.23 g
of acetic acid. Then, 3.23 ml of cooled dimethylamine
(25% in H2O) was added. To the resulting cooled solution
was added 2.7 g 115.2 mmoles) of compound of the formula
XVII using about l.S-2.0 ml of tetrahydrofuran as trans-
fer solvent. The reaction was warmed to room temperature
and stirred overnight. Then, 40 ml of 10% NaOH was
added whereupon a precipitate formed. After stirring,
the precipitate was filtered, washed with H2O and air
dried to yield 3.26 g of compound of the formula XVIII.
C. XVIII
OCH20CH3 OCH20CH3
OH + ~ N~2
H H
(XIX) (XX)
Under nitrogen atmosphere, 4.62 g of KCN was com-
bined with 10 ml H2O and stirred into solution. Then
30 ml of ethanol and 3.26 g (14 mmoles) of compound
XVIII ~ere added and the mixture was stirred and heated
-
21 941 03
-54-
to reflux for about 65 hours. The reaction was cooled
and concentrated. The resulting precipitate comprisinq
compound XX was filtered, washed with H2O and air
dried. The aqueous layer was extracted with 4 x 15 ml
dichloromethane, sa~ing the organic extracts. The
aqueous layer then was overlayed with 50 ml ethyl acetate
and acidified to pH 2. The ethyl acetate layer was
separated, degassed by bubbling nitrogen through the
ethyl acetate layer, dried and concentrated to a solid
which was triturated with diisopropyl ether to yield
1.03 g of compound XIX as an off-white solid. Then,
all extracts from the basic aqueous layer were combined,
dried, combined with the filtrates from above and con-
centrated to yield 0.4 g of compound XX as a solid.
D. XX ~ XIX
Under nitrogen atmosphere, 1.6 g of compound XX
was combined with 7.5 ml of ethanol, 30 ml H2O and
1.3 g KOH. The mixture was heated to reflux and
stirred overnight. Then, the reaction was cooled,
extracted with 2 x 15 ml ethyl acetate, overlayed with
fresh ethyl acetate and acidified to pH 2. The
combined ethyl acetate layers were dried and
concentrated. The resulting solid was triturated with
IPE to yield 1.1 g of compound XIX.
E. OCH2OCH3
XIX ~ ~ OCH2CH=CH2
BOC
(XXI)
In 10 ml H2O was dissolved 1.44 g (4.25 mmoles)
TsAHSO4. Then, 714 mg (8.5 mmoles) of NaHC03 was
added. After foaming subsided, l.0 g (4.25 mmoles) of
-
_55_ 21 941 03
compound of formula XIX was added and the mixture was
stirred for one minute before 40 ml of chloroform was
added. The reaction was stirred for 5 minutes, the
chloroform layer was separated and then the aqueous
mixture was extracted with 1 x 15 ml chloroform. The
combined chloroform extracts were dried, concentrated
and chased with 2 x 30 ml of acetone. Then, the product
was redissolved in 30 ml of acetone under nitrogen
atmosphere, 0.37 ml (4.25 mmoles) of allyl bromide was
added and the reaction was stirred for 2 hours. The
mixture was concentrated and chromatographed using
ethyl acetate as the eluent. The product was concen-
trated to yield 1.1 g of an oil. All of the product
was combined with 25 ml dichloromethane and 0.98 g (4.5
mmoles) of di-t-butyldicarbonate and 51 mg (0.425 mmoles)
4-(N,N-dimethylamino)pyridine were added. The reaction
was stirred overnight. Then, the reaction mixture was
concentrated and chromatographed using 9:1 hexane/ethyl
acetate to yield, after concentration, 1.33 g of the
compound of formula XXI as a viscous oil.
F. OCH2OCH3
XXI
BOC
(XXII)
. In 2 ml of methanol was dissolved 64 mg (0.17
mmoles) of compound of the formula XXI. To that solution
was added 1.7 ml of 0.1 NaOH and the resulting reaction
Z5 mixture was stirred overnight. The reaction then was
acidified, dried, extracted with ethyl acetate and con-
centrated. NMR analysis showed all of the allyl ester
of compound XXI had been removed but some trans ester-
ification had occurred and some methyl ester ~as present.
-56- 21 941 03
Therefore, the product was combined with the remaining
amount of compound XXI (approx. 1.26 g), dissolved in
S ml of tetrahydrofuran and cooled to 0~ C. Then,
3.8 ml of lN NaOH was added, the reaction was stirred
for 5 minutes at 0~ C. then allowed to warm to room
temperature. The reaction resulted in two phases which
required the addition of 5 ml methanol to eliminate the
phases. The reaction was stirred overnight. Then, the
tetrahydrofuran and methanol were stripped off and the
remaining aqueous solution was overlayed with 50 ml
ethyl acetate and acidified to pH 2. The ethyl acetate
layer was separated, washed with l x 20 ml H2O, once
with brine, dried with Na2SO4, filtered and concentrated
to a gum which crystallized when triturated with hexane
to yield 1.08 g of white solid. The solid was chromato-
graphed using 70:30 ethyl acetate/hexane and the clean
product cuts were combined, concentrated and crystallized
from ethyl acetate/hexane to yield 0.884 g of compound
of the formula XXII.
G. XXII
BOC
H ~ N ~'~ ~N~'~'~N~ N~_~--~_,N-BOC
H BOC BOC
(X)
OCH20CH3 H H B(~C BOC
~N ,N N ~ N ~N N -BOC
BOC
(XXIII )
_ 2194103
Under nitrogen atmosphere, 271 mg (0.81 mmoles) of
compound of the formula XXII was dissolved with stirring
in 4 ml of anhydrous tetrahydrofuran. Then, 93 mg
(O.81 mmoles) of N-hydroxysucc~nimide and 167 mg ~0.81
mmoles) of dicyclohexvlcarbodiimide were added and the
mixture was stirred. After one hour and fif~y minute~,
O.58 g (0.8 mmoles) of compound of the formula X, pre-
pared according to the procedure described in Example 11,
in 30 ml dichloromethane was added and the m~xture
stirred over the weekend Then, the dicyclohexylurea
was filtered off, and the mixture was extracted with 2
x 3 ml 1N NaOH, dried over R2C03 and concentrated. The
produst then was chromatographed on 100 9 silica gel
using 9:1 dichloromethane/methanol to elute everything
but the desired product then with 90:10:1 dichlorometh-
ane/methanol/isopropvlamine to elute the product. The
product containing fractions were concentrated, chased
several times with dichloromethane and stripped of
solvent to yield 349 mg of compound of the formula
XXIII as a white foam.
Alternatively, and preferably, a 50 ml solution
containing 0.480 g (1.43 mmoles) of compound of the
formula XXII in dichloromethane was treated with 0.165 g
(1.43 mmoles) of N-hydroxysuccinimide followed by 0.294 g
(1.43 mmoles) of dicyclohexylcarbodiimide and allowed to
stir for S hours. The reaction mixture was filtered and
the filtrate containing crude hydroxysuccinimide was added
drop~ise over a 20 minute period to a 100 ml solution-of
1.03 g (1.43 mmoles) of compound of the formula X, prepared
according to the procedure described in Example 11, in
dichloromethane. The reaction was stirred for 7~ hours.
Then, the crude reaction mixture was washed twice with
-58- 2 1 9 4 1 0 3
20 ml of lN NaOH, dried over K2CO3, filtered and concen-
trated. The product was then chromatographed on silica
gel using 4:1 dichloromethane/methanol followed by 9:1:1
dichloromethane/methanol/diisopropylamine to afford 912 mg
of compound of the formula XXIII.
H. XXIII
OCH2OCH3 H . BOC BOC
N N~,_~_~N~,-~_, ~BOC N-BOC
BOC
(XXIV)
Under a nitrogen atmosphere, indoleacetamide XXIII
(900 mg, 0.87 mm~l) was dissolved in 40 ml of dichloro-
methane. To this solution was added 2-(phenylsolfonyl)-
3-phenyloxaziridine (500 mg, 1.92 mmol). Progress of
the reaction was monitored by thin layer chromatography.
After 1 hour the reaction mixture was concentrated in
vacuo to give predominantly crude nitrone which was
dissolved in 35 ml of acetic acid. A large excess of
sodium cvanoborohydride (1.00 g, 15.9 mmol) was added
and the reaction was allowed to stir for about 2 hours.
The reaction was concentrated in vacuo, taken up in di-
chloromethane (50 ml), washed with pH 7 buffer (1 x
50 ml) adjusting the pH to 7 with lN NaOH. The organic
layer was washed again with pH 7 buffer (1 x 50 ml),
dried over potassium carbonate, and concentrated in
vacuo to generate crude product which was chromato-
graphed on silica gel using 95:5 ethyl acetate/methanol
to afford 612 mg (67%) of XXIV as a white foam.
sg 21 941 03
I. XXIV
OH H OH H H
U N~ ~N~ ~N\ "~_~N\~ ~H NH2
- (XXV)
By alternately pulling a vacuum and bleeding with
argon three times, 3 ml of trifluoroacetic acid was
degassed. Then, the flask was wrapped in aluminum foil
and 98 mg of compound XXIV was added with the use of
about 2.5 ml dichloromethane as a transferring agent.
After 45 minutes, the mixture was concentrated to yield
an oil which, upon trituration with diethyl ether,
yielded 88 mg of compound XXV as a white solid which
was stored at -80~ C.
Alternatively, and preferably, to a saturated
100 ml dioxane-HCl solution, purged with argon, was
added, over 1.5 minutes, 0.520 g (0.495 mmoles) of
compound of formula XXIV, prepared according to the
procedure described in part H, above, in 10 ml of
dioxane. A precipitate formed immediately and the
reaction was stirred for 1 hour. Then, diethylether
was added and, after 5 minutes, the solution was
filtered. The resultant solids were washed with diethyl-
ether, dried under nitrogen atmosphere and then under
reduced pressure to afford 345 mg of the acid of formula
XXIV' which could be stored at -78~C. Then, to an
aqueous solution, purged with argon, was added 150 mg
(0.205 mmoles) of the acid of formula XXIV'. The reac-
tion was monitored by HPLC. Upon completion of the
reaction after about 7 hours, the reaction mixture was
freeze dried to afford 103 mg of compound of the
formula XXV as its hydrochloride salt.
-60- 21 941 03
EXAMPLE 13
Benzamide, N-(20-amino-~-hydroxy-4,8,12,17-
tetraazeicos-1-yl)-~-hydroxy
synthesis of the title compound, ascertained to be
comprised by fraction A' as described in Example 2 was
accomplished as desc~ibed below.
A. O
Il 11
~ OH ~ OCH2CH=CH2
HO NO
(XXVI)
In a 400 ml beaker, 6.78 g (20 mmoles) of tetra~utyl-
ammonium hydrogen sulfate was combined with 60 ml H2O
and stirred into solution. Then, 3.36 g (40 mmoles) of
NaHCO3 was added as foaming would allow followed by the
addition of 40 ml of a solution containing 2.76 g (20
mmoles) p-hydroxybenzoic acid, 0.8 g NaOH and 40 ml
H2O. The reaction mixture was stirred for 5 minutes
lS and then 200 ml of dichloromethane was added. The
mixture was stirred for another S minutes and the layers
were allowed to separate. The aqueous layer was ex-
tracted twice with 50 ml of dichloromethane. The com-
bined dichloromethane extracts were dried o~er Na2SO4,
concentrated, chased twice with 75 ml acetone and
redissolved in 50 ml acetone. Then, 1.9 ml (22 mmoles~
of allyl bromide were added and the mixture was stirred.
The reaction mixture was then concentrated, taken up in
100 ml ethylacetate, washed twice with 30 ml H2O, once
with 50 ml saturated bicarbonate, once with 30 ml H2O,
once with brine, once with H2O and once with brine.
The resulting product was dried and concentrated to
yield an oil. The oil was triturated with 50 ml
petroieum ether for one hour. Then, the mixture was
21941Q3
-61-
filtered, washed well with petroleum ether and air
dried to yield 1.7 g of compound of the formula XXVI,
above.
B. ~l
~ OCH2CH=CH2
XXVI ~ ~
C~30CH20
(XXVII)
Under a nitrogen atmosphere, 1.7 g (9.55 mmoles)
of compound of the formula XXVI, prepared as described
in part A, above, was combined with 50 ml of Aldrich
dry DMF and stirred into solution. Then, O . 38 g (9.55
mmoles) of NaH as a 60% oil dispersion was added and
the reaction was stirred overnight. Then, 0.76 ml of
chloromethylmethyl ether (Aldrich) was added whereupon
a precipitate formed immediately. The reaction mixture
was stirred for 18 hours. The reaction was quenched in
200 ml ethylacetate/100 ml H2O. The ethylacetate layer
was separated, washed 3 times with 50 ml H2O, once with
50 ml lN NaOH, once with 50 ml H2O and once with brine.
The resulting solution was dried, concentrated and
chromatographed on silica gel using 4:1
hexane/ethylacetate to yield 1.63 g of compound of
the formula XXVII, above.
C. 1~l
~ OH
XXVII ~ ~
CH30C~20
(XXVIII)
In 75 ml tetrahydrofuran was dissolved 1.62 g (7.3
mmoles) of compound of the formula XXVII, prepared as
- 21 941 03
-62-
described in part B, above. Then a solution containing
O.4 g (10 mmoles) of NaOH in 15 ml of ~2~ was added
resulting in the formation of two layers. ~ethanol was
added until a homogeneous mixture was obtained. The
S reaction was then 8tirred overnight. The tetrahydro-
furan and methanol were stripped off in vacuo and the
remaining aqueous solution was extracted tw~ce with
15 ml ethylacetate. Then, the aqueous layer was over-
layed with 50 ml of fresh ethylacetate and acidified to
pH 2.5 with 6N HCl. Then ethylacetate layer was
separated, washed once with 10 ml H2O and once with
25 ml brine. The resulting solution was concentrated
to yield solids which were triturated with hexane,
filtered and air dried to yield 1.2 g of compound of
formula XXVIII, above.
D. X + XXVIII
~OC
~N~ ~~ N N- BOC
~ ~ BOC BOC
OCH 2~CH 3
(XXXII)
A 20 ml dichloromethane solution contalning 0.127 g
(0.7 mmoles) of compound of the formula XXVIII, prepared
as described in Part C, above, was treated with 0.081 g
(0.7 mmoles) of N-hydroxysuccinimide followed by 0.144 g
(0.7 mmoles) of dicyclohexylcarbodiimide and allowed to
stir for 4 hours. Then, the reaction mixture was
filtered and the filtrate containing crude hydroxy-
succinimide was added dropwise over a 20 minute periodto a 80 ml dichloromethane solution containing 0.50 g
(0.7 mmoles) of compound of the formula X, prepared
according to the procedure described in Example 11,
21 941 03
-63-
Parts A-I, above. The xeaction was then stirred for 17
hours. The reaction mixture was then washed with lN
NaOH (2 x 20 ml), dried over potassium carbonate,
filtered and concentrated. The concentrate was chromato-
graphed on silica gel using 9:1 dichloromethane~methanolfollowed bv 9:1:0.25 dichloromethane/methanol/d~iso-
~ropylamine to yield 370 mg of compound of the fonmula
XXXII, above.
E. XXXII
BOC
~~N~ N~ N~~N ~N-Boc
~ H 0H BOC BOC
OC~l20C'~
(XXXIV)
Under a nitrogen atmosphere, 0.335 g (0.38 mmoles)
of compound of the formula XXXII, prepared according to
the procedure described ln Part D, above, was dissolved
in 30 ml of dichloromethane. To this solution was
added 0.2~0 g (0.88 mmoles) of 2-(sulfonylphenyl)-3-
phellyl-oxaziridine. After 0.5 hour the reaction was
collcentrated in vacuo and the concentrate was dissolved
in 15 ml of acetic acid. A large excess of cyanoboro-
hydride (.100 g, 1.6 mmoles) was added and the reaction
was stirred for 2 hours. Then, the reaction was con-
centrated in vacuo, taken up in 50 ml of dichloro-
methane, washed once with 50 ml of pll 7 buffer, adjust-
ing the pll to 7 with lN NaOH. The organic layer was
~ashed again with 50 ml of pl~ 7 buffer, dried over
potassium carbonate and concentrated in vacuo. The
concelltrate was chromatographed on silica gel using
50:50 acetone/hexane to yield 253 mg of compound of the
formula XXXIV, above
-64- 2194103
F. (XXXIV)
--~N~ N~ N~~H NH2
H O~ H
HO
- (XXXVI)
Under an argon atmosphere at room temperature were
combined 0.2C g ~0.223 mmoleQ1 of compound of the formula
XXXIV, prepared according to the procedure described in
Part E, above, and 5 ml of trifluoroacetic acid. The
reaction was stirred for 1.5 hours. An additional 2 ml
of trifluor~acetic acid was added to wash the walls of the
flask. The reaction was allowed to proceed for an
additional l.S hours. Then, the reaction wa~ concen-
trated in vacuo, triturated with ethyl ether, filtered
and dried under a nitrogen atmosphere to afford 218 mg
of compound of the formula XXXVI, the title compound of
this Example.
EXAMP~E 14
Benzamide, N- (20-amino-4-hydroxy-4,8,12,17-
tetraazeicos-l-yl)-2,5-dihydroxy
Synthesis of the title compound, ascertained to be
comprised by fraction Al as described in Example 3, was
accomplished as described below.
A- O~ O O~ O
1 11 I ll
(~\01~ ~\ OCH2CH-CH2
OH OH
(XXIX1
64680-788D
- 21 941 03
-65-
Employing the procedure described in Example 12,
Part A, but 3tarting with 3.08 g (20 mmoles) of
2,5-dihydroxybenzoic acid yielded 3.0 g of compound of
the formula XXIX, a~ove.
B. OC~2OC~3
~\OC~2C~=C~2
XXIX ~ ~
OC~2OCH3
(XXX )
Under nitrogen atmosphere, 3.0 g (15.5 mmoles) of
compound of the formula XXIX was combined with 50 ml of
Aldrich dry DMF and stirred into solution. Then, 1.24 g
(31 mmoles~ of NaH as a 60% oil dispersion was added
and the mixture was stirred. After 3 hours, 2.5 ml (33
mmoles) of chloromethylmethyl ether was added and the
reaction ~as stirred for 18 hours. The reaction mixture
was then added to 300 ml ethylacetate and 100 ml H2O.
The ethylacetate layer was then separated and washed 3
lS times with 75 ml H2O, twice with 75 ml lN NaO~, twice
with 50 ml H2O and once with brine. The resulting
solution was dried and concentrated to yield 2.9 g of
an oil which was chromatographed on silica gel using
4:1 hexane/ethylacetate to yield two fractions, the
second fraction of which contained 1.16 5 of compound
of the formula XXX, above.
C. OCH20CH3
I R
~ OH
XXX ~ ~
OCH20C~3
(XXXI)
21 941 03
-66-
To l0 ml of tetrahydrofuran was added l.16 g (4.1
mmoles) of compound of the formula XXX, above. Then,
O.24 g (6 mmoles) of NaOH in 10 ml H20 was added which
resulted in the formation of two phases. Methanol was
added t~ the mixture until one phase was obtained and
then the mixture was stirred for 18 hours. The
solvents were then removed and the remaining aqueous
solution was extracted twice with 15 ml of ethylacetate.
The aqueous layer ~as then overlayed with 35 ml of
fresh ethylacetate and the pH was adjusted to 2.5 with
6N HCl. The ethylacetate layer was then separated,
washed once with 10 ml ~2~' and once with brine, dried
and concentrated to yield 1.0 g of compound of the
formula XXXI, above, as an oil.
15 D. X + XXXI
OCH2OCH3
BOC
~~~~'~~'~~~'~~N'--~_--~ " N~ OC
H H . BOC
OCH20CH3
(XXXIII)
Employing the procedure described in Example 13,
Part D, and using 0.17 g (0.7 mmoles) of compound of
the formula XXXI, prepared as described in Part C,
above, 0.081 g (0.7 mmoles) N-hydrox~succinimide,
0.144 g (0.7 ~moles) dicyclohexylcarbodii.mide and
0.50 g (0.7 mmoles) of compound of the for~ula X,
prepared according to the procedure described in
Example 11, Parts A-I, yielded, after chromatographv,
370 mg of compound of the formula XXXIII, above.
-67- 2194103
E. XXXIII
~Cff2~CH3 BOC
N~ ~N~--~--~--~" N~ N-30c
OR BOC BOC
OCH20Ct~3
- ( XXXV)
Employing the procedure described in Example 13,
Part E, and using O.308 g (0.33 mmoles) of compound of
the formula XXXIII, prepared according to the procedure
described in Part D, above, and 0.250 g (0.96 mmoles)
of 2-(sulfonylphenyl)-3-phenyl-oxaziridine yielded,
after silica gel chromatography using ethylacetate
followed by 50:50 acetone/hexane, 210 mg of compound of
the formula XXXV, above.
F. XXXV
N'~~--~~~N'--~--' " N~--~--~--~"'~'"'~
OH H H
OH
(XXXVII)
Employing the procedure described in Example 13,
Part F, and using 0.100 g tO.104 mmoles) of compound of
the formula XXXV, prepared according to the procedure
described in Part E, above, yielded 108 mg of compound
of the formula XXXVII, the title compound of this
Example.
-68- 21 ~41 03
EXAMPLE 1 5
lH-Indole-3-acetamide, N-(16-amino-4-hydroxy-
4,8,13-triazahexadec-1-yl)-4-hydroxy
Synthesis of the title compound, ascertained to be
comprised by fraction A2 as described in Example 3, was
accomplished as descrlbed below.
. BOC
VI ~ H2N'--~--''~N~'~--~'~, ' N-BOC
BOC
(XXXVIII)
Employing the procedure described in Example 11,
Part D, and using 1.15 g (2,07 mmoles) of compound of
the formula vI, prepared as described in Example 11,
Parts.A-E, yielded 1.10 g of crude compound of the
formula XXXVIII, above.
~ XXXVI I I + XXI I
OCH 2~CH 3 H H BOC
~N N ~NWN ~ BOC H- BOC
BOC
(XXX IX)
Employing the alternate procedure described in
Example 12, Part G, and using a dichloromethane solution
115 ml) containing 0.228 g (G.8 mmoles) of compound of
the formula XXII, prepared as described in Example 12,
Parts A to F, above, 0.380 g ~6.8 mmoles) of compound
of the formula xxXVIII, prepared as described in Part A,
above, 0.078 g (6.8 mmoles) of N-hydroxysuccinimide and
0.140 g (6.8 mmoles) of dicyclohexylcarbodiimide afforded
0.407 g ~f compound of the formula XXXIX after silica
-69- 21 941 03
gel chromatography using 9:1 dichloromethane/methanol
followed by 9:1:0S dichloromethane/methanol/diisopropyl-
amine.
~ XXX 1 X
OCH20CH3 H ~ BOC
N~,~_~N\-~N~-~'~-~~~~~N-Boc
N~ ~ 80C H
BOC
(XL)
Employing the procedure described in Example 12,
Part H, and using 0.350 g (0.4 mmoles) of compound of
the formula XXXIX and O.230 g (O.a8 mmoles) of
2-(sulfonylphenyl)-3-phenyloxziridine gave 0.274 g of
compound of the formula XL, above, after silica gel
chromatography using 50:50 acetone/hexane. The product
contained a minor amount of phenylsulfonamide and
therefore was further purified on silica gel usinq
ethylacetate followed by SO:SO acetone/he.Yane.
XL
OH H OH H
~N N ~ N ~ N ~ --N H
(XLI)
Employing the alternate procedu e of Example 12,
Part I, and using 0.166 g (0.186 mmoles) of compoun~ of
- 21 941 03
-70-
the formula XL, prepared according to the procedure
described in Part C, above, provided after dioxane/HCl
treatment crude acid. The acid of formula XL' was
dissolved in water for 8 hours, followed by freeze
drying to yield 88 mg of compound of the formula XLI.
' 2194103
-71-
PREPARATION A
Br~ N-BOC
H
Under nitrogen atmosphere, 34.5 g (157.6 mmoles)
of 3-bromopropylamine-HBr in 600 ml of N,N-dimethylform-
amide was stirred. To that solution was added 34.4 g
(157.6 mmoles) of di-t-butyldicarbonate followed by
32.3 ml (236 mmoles) triethylamine. A precipitate
formed immediately. The reaction was stirred overnight.
The reaction mixture was then diluted to 1.5 liters
with ethylacetate, washed once with 500 ml of lN HCl,
three times with 500 ml water, once with brine and
dried over Na2SO4. After concentration, the product
was chromatographed on 800 g silica gel using 4:1
hexane/ethylacetate and the fractions were monitored by
thin layer chromatography (KMNO4/I2). The fractions
containing the product were combined, concentrated in
vacuo, chased twice with 50 ml dichloromethane and
purged with high vacuum to yield 25.8 g of the product
of this Preparation.
